The goal of this project is to understand the molecular mechanisms of protein synthesis based on determination of x-ray crystal structures of functional complexes of ribosomes. Now that the structure of the ribosome is known at high resolution, the challenge is to determine how its structure changes in different functional states during protein synthesis. One major unsolved problem is the mechanism of coupled translocation of mRNA and tRNA, which involves large-scale rearrangements of ribosome structure, and is catalyzed by elongation factor EF-G. Complexes of ribosomes bound with EF-G will be trapped in intermediate states of translocation by use of GTP analogs, antibiotics and directed mutations in the ribosome and EF-G for crystallization and structure determination. A second project is to understand the mechanisms of termination of protein synthesis, which depends on the class I release factors RF1 and RF2, and requires participation of release factor RF3 and GTP to then release RF1 and RF2. How RF3 causes release of the type I factors is unknown, but is believed to involve changes in ribosome conformation similar to those seen for EF-G during translocation. RF3 will be bound to termination complexes containing RF1 or RF2 bound in response to a stop codon, and crystallized for structure determination. A third poorly understood mechanism is how the ribosome unwinds structured mRNAs with its endogenous helicase activity. This problem will be addressed by determining the structures of ribosomes containing mRNAs with double-helical and pseudoknot structures stalled in their helicase active sites. The proposed research is of strong clinical relevance because bacterial ribosomes are a major target for numerous antibiotics. Elucidation of the structures of functional complexes of bacterial ribosomes will provide a rigorous basis for development of novel antibiotics to address the crisis of emerging drug-resistant strains of bacterial pathogens. Studying the mechanism of action of the ribosomal helicase will help to understand how retroviruses, including HIV, use pseudoknots to create programmed frame-shifting during their infectious cycle, providing clues to the design of anti-retroviral therapeutics.